Exploring the Utility of Multiplex Infectious Disease Panel Testing for Diagnosis of Infection in Different Body Sites: A Joint Report of the Association for Molecular Pathology, American Society for Microbiology, Infectious Diseases Society of America, and Pan American Society for Clinical Virology.

The use of clinical molecular diagnostic methods for detecting microbial pathogens continues to expand and, in some cases, supplant conventional identification methods in various scenarios. Analytical and clinical benefits of multiplex molecular panels for the detection of respiratory pathogens have been demonstrated in various studies. The use of these panels in managing different patient populations has been incorporated into clinical guidance documents. The Association for Molecular Pathology's Infectious Diseases Multiplex Working Group conducted a review of the current benefits and challenges to using multiplex PCR for the detection of pathogens from gastrointestinal tract, central nervous system, lower respiratory tract, and joint specimens. The Working Group also discusses future directions and novel approaches to detection of pathogens in alternate specimen types, and outlines challenges associated with implementation of these multiplex PCR panels.

PivNG primers and probes set used in the cobas omni Utility Channel is a reliable supplemental test for detection of Neisseria gonorrhoeae in oropharyngeal, urogenital and rectal specimens collected in cobas PCR Media.

OBJECTIVE: To evaluate the clinical performance of the novel PivNG primers and probes set (PivNG test) used in the cobas omni Utility Channel for supplemental testing of Neisseria gonorrhoeae (NG). METHODS: Oropharyngeal, urogenital and rectal samples were self-collected during routine testing at Barts Health sexual health clinics, London, UK. Samples were tested by the cobas CT/NG test and PivNG cobas omni Utility Channel test on cobas 6800/8800 Systems. Supplemental testing was carried out with the Xpert CT/NG test. PivNG overall percent agreements, positive percent agreements (PPAs)/negative percent agreements (NPAs) and positive/negative predictive values were calculated for each sample type. Microscopy and/or culture data were included for a randomised subset of concordant/discordant results, and a composite reference standard (cobas CT/NG, Xpert CT/NG and culture results) adjusted for partial verification bias was used to determine PivNG PPA and NPA. RESULTS: Of 447 evaluable samples with valid results from all three assays (cobas CT/NG, PivNG and Xpert CT/NG), 288 (64.4%) were NG-positive by both PivNG and cobas CT/NG; 117 (26.2%) were NG-negative in both tests; and 42 (9.4%) had discordant results (with NG-negative supplementary Xpert) CT/NG results in 40/42 instances). Of 19 PivNG/Xpert CT/NG-discordant samples, 11 were confirmed NG-positive by microscopy and/or culture results. PivNG PPA and NPA were 100% and 91% for oropharyngeal swabs, 100% and 100% for vaginal swabs, 100% and 100% for male urine samples, and 100% and 97% for rectal swabs, respectively, compared with the partially adjusted composite reference standard. CONCLUSIONS: PivNG is a reliable supplementary test with high sensitivity for confirming NG infection when used in conjunction with the cobas CT/NG test and samples collected in cobas PCR Media. Moreover, the PivNG test offers a convenient, high-throughput solution for supplemental NG testing of various sample types, with the potential to reduce the number of indeterminate reports.

The prevalence of Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) at testing centers in Belgium, Germany, Spain, and the UK using the cobas TV/MG molecular assay.

Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) can lead to long-term sequelae in males and females; however, global prevalence data vary between geographical regions, as these sexually transmitted infections are not included in routine screening. The objective of this study was to use the cobas® TV/MG assay to assess the point prevalence of TV and MG in specimens from men and women over a broad European geographical area. Urine, vaginal, endocervical, and rectal samples were collected from patients aged ≥ 18 years receiving Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (NG) screening as per local standard of care at sites in Belgium, Germany, Spain, and the UK (Wales). Remnant samples were assessed using the cobas TV/MG assay. Analysis of 2795 samples showed that MG prevalence varied slightly across female sample types (range: 1.7-5.8%; p = 0.0042). MG prevalence was higher in male rectal samples (12.5%) than in male urine samples (3.9%; p < 0.0001). TV prevalence was low in male (0.8%; 12/1535) and female (1.3%; 16/1260) samples across all sites. Co-infection of TV/MG with CT or NG was 10.0% (19/190) and 9.6% (7/73), respectively, in both male and female samples. MG and TV prevalence rates were comparable to the published literature in Europe. MG prevalence was highest in male rectal samples; as rectal testing is an off-label use of the cobas TV/MG assay, the clinical utility of this assay for rectal testing should be further investigated.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae with the cobas CT/NG v2.0 test: performance compared with the BD ProbeTec CT Qx and GC Qx amplified DNA and Aptima AC2 assays.

OBJECTIVES: Infections due to Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most common bacterial sexually transmitted infections worldwide, most of which are asymptomatic. Detection of infection using a variety of specimen types in symptomatic and asymptomatic subjects is important to effectively combat CT/NG infections. The performance of the cobas CT/NG v2.0 test was assessed for urogenital swabs, urine and cervical cytology samples collected in PreservCyt Solution from 5266 symptomatic and asymptomatic women (including 202 who were pregnant), and urine from 738 men. METHODS: Sensitivity and specificity were estimated compared with a patient infected status determined using two US Food and Drug Administration-cleared nucleic acid amplification tests. RESULTS: Among 6004 participants, 487 CT (8.1%) and 159 NG (2.6%) infections were identified. Sensitivity estimates for CT for women ranged from 91.2% to 97.6% depending on specimen type, and the estimate for male urine specimens was 98.4%. Specificity for CT ranged from 99.2% to 99.7%. Sensitivity estimates for NG ranged from 95.6% to 100.0% for women, and the estimate for men was 100.0%. Specificity for NG ranged from 99.3% to 100.0%. CONCLUSIONS: The cobas CT/NG v2.0 test performs well using urogenital swabs, urine and cervical samples collected in PreservCyt solution.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Performance of the cobas MRSA/SA Test for Simultaneous Detection of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus From Nasal Swabs.

OBJECTIVES: Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA) infections are continuing problems. Rapidly determining the MRSA colonization status of a patient facilitates practice to reduce spread of MRSA clinical disease. Sensitive detection of all SA prior to surgery, followed by decolonization, can significantly reduce postoperative infection from this pathogen. Our goal was to validate a new automated assay for this testing. METHODS: We compared performance of the cobas MRSA/SA Test on the cobas 4800 System to direct and enriched chromogenic culture using nasal swabs collected from patients at six United States sites. RESULTS: Compared to direct and enriched culture, the sensitivity for MRSA and SA was 93.1% and 93.9%, and the specificity was 97.5% and 94.2%, respectively. After discrepancy analysis, the sensitivity for MRSA and SA was 97.1% and 98.6%, and the specificity was 98.3% and 95.5%, respectively. Compared to direct culture, sensitivity for detecting any SA was 99.6%. CONCLUSIONS: The cobas MRSA/SA Test is an effective tool to simultaneously perform surveillance testing for nasal colonization of both MRSA and MSSA.

Evaluation of the cobas Cdiff Test for Detection of Toxigenic Clostridium difficile in Stool Samples.

Nucleic acid amplification tests (NAATs) are reliable tools for the detection of toxigenic Clostridium difficile from unformed (liquid or soft) stool samples. The objective of this study was to evaluate performance of the cobas Cdiff test on the cobas 4800 system using prospectively collected stool specimens from patients suspected of having C. difficile infection (CDI). The performance of the cobas Cdiff test was compared to the results of combined direct and broth-enriched toxigenic culture methods in a large, multicenter clinical trial. Additional discrepancy analysis was performed by using the Xpert C. difficile Epi test. Sample storage was evaluated by using contrived and fresh samples before and after storage at -20°C. Testing was performed on samples from 683 subjects (306 males and 377 females); 113 (16.5%) of 683 subjects were positive for toxigenic C. difficile by direct toxigenic culture, and 141 of 682 subjects were positive by using the combined direct and enriched toxigenic culture method (reference method), for a prevalence rate of 20.7%. The sensitivity and specificity of the cobas Cdiff test compared to the combined direct and enriched culture method were 92.9% (131/141; 95% confidence interval [CI], 87.4% to 96.1%) and 98.7% (534/541; 95% CI, 97.4% to 99.4%), respectively. Discrepancy analysis using results for retested samples from a second NAAT (Xpert C. difficile/Epi test; Cepheid, Sunnyvale, CA) found no false-negative and 4 false-positive cobas Cdiff test results. There was no difference in positive and negative results in comparisons of fresh and stored samples. These results support the use of the cobas Cdiff test as a robust aid in the diagnosis of CDI.

Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/µL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays.

Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity.

Assessment of Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Bacterial and Yeast Identification.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of clinical bacterial and yeast isolates. However, data describing the reproducibility of MALDI-TOF MS for microbial identification are scarce. In this study, we show that MALDI-TOF MS-based microbial identification is highly reproducible and can tolerate numerous variables, including differences in testing environments, instruments, operators, reagent lots, and sample positioning patterns. Finally, we reveal that samples of bacterial and yeast isolates prepared for MALDI-TOF MS identification can be repeatedly analyzed without compromising organism identification.

Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria.

The VITEK MS v2.0 MALDI-TOF mass spectrometry system's performance in identifying fastidious gram-negative bacteria was evaluated in a multicenter study. Compared with the reference method (DNA sequencing), the VITEK MS system provided an accurate, species-level identification for 96% of 226 isolates; an additional 1% were accurately identified to the genus level.

Multicenter evaluation of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Gram-positive aerobic bacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

Multicenter study evaluating the Vitek MS system for identification of medically important yeasts.

The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Los Angeles County.

Data on Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH) in adults in the United States remain very limited. A cluster of four cases of EBV-HLH was observed in a 4-month period at a tertiary center in Los Angeles County (LA County) and the clinical and molecular characteristics identified in these cases are being described. EBV typing, immunophenotypic and molecular genetic studies were performed. Diagnostic criteria that may be used to identify EBV as a cause of HLH in adults are also being suggested. Finally, the crude incidence rate for HLH in LA County was determined and was compared to the worldwide crude incidence rate for HLH. The cases each occurred in young male adult residents of California and were associated with evidence of EBV reactivation and ferritin levels of >20,000 µg/L. A higher rate of cases of EBV-HLH in 2010 was found at UCLA Medical Center than for 2007-2009 (4.9/10,000 hospital discharges vs. 0.14/10,000 hospital discharges, respectively; P = 0.0017). The cases were associated with EBV type 1, and the insertion of the codon CTC (leucine) was found in numerous of the EBNA-2 gene sequences. The annual incidence of secondary, non-familial HLH was estimated to be 0.9 cases per million persons >15 years of age in LA County. Although EBV-HLH is a rare disease, the incidence in adults in Western countries may be underestimated.

Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory.

Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.

Target-specific capture enhances sensitivity of electrochemical detection of bacterial pathogens.

We report the concentration and purification of bacterial 16S rRNA by the use of a biotinylated DNA target-specific capture (TSC) probe. For both cultivated bacterial and urine specimens from urinary tract infection patients, TSC resulted in a 5- to 8-fold improvement in the sensitivity of bacterial detection in a 16S rRNA electrochemical sensor assay.

Combination therapy with daptomycin, linezolid, and rifampin as treatment option for MRSA meningitis and bacteremia.

Methicillin-resistant Staphylococcus aureus (MRSA) meningitis is associated with a high mortality rate. Treatment is challenging in patients with allergy to vancomycin. Herein, we describe a case of MRSA bacteremia secondary to medical device infection with MRSA that was complicated by MRSA meningitis. This case provides evidence for a possible role of combination therapy of daptomycin, linezolid, and rifampin in cases of MRSA meningitis and bacteremia.